Signal-sustained imaging of mitophagy with an Enzyme-Activatable Metabolic Lipid-Labeling Probe

Imaging of mitophagy is of significance as aberrant mitophagy is engaged in multiple diseases. Mitophagy has been imaged with synthetic or biotic pH sensors by reporting pH acidification en route delivery into lysosomes. To circumvent uncertainty of acidity-dependent signals, we herein report an enzyme-activatable probe covalently attached on mitochondrial inner membrane (ECAM) for signal-persist mitophagy imaging. ECAM is operated via ΔΨm-driven accumulation of Mito-proGreen in mitochondria and covalent linking of the trapped probe with azidophospholipids metabolically incorporated into the mitochondrial inner membrane. Upon mitophagy, ECAM is delivered into lysosomes and hydrolyzed by LNPEP/leucyl aminopeptidase, yielding turn-on green fluorescence that is immune to lysosomal acidity changes and stably retained in fixed cells. With ECAM, phorbol-12-myristate-13-acetate (PMA) was identified as a highly potent inducer of mitophagy. Overcoming signal susceptibility of pH probes and liability of ΔΨm probes to dissipation from stressed mitochondria, ECAM offers an attractive tool to study mitophagy and mitophagy-inducing therapeutic agents.

线粒体自噬（mitophagy）成像具有重要研究意义，异常线粒体自噬与多种疾病的发生发展紧密相关。既往研究通过合成或生物源性pH传感器，追踪其在转运至溶酶体（lysosome）过程中的pH酸化信号，实现线粒体自噬成像。为规避酸度依赖型信号带来的检测不确定性，本文报道一种共价锚定在线粒体内膜的酶激活型探针（ECAM），可实现信号稳定持久的线粒体自噬成像。
ECAM的工作机制基于线粒体膜电位（ΔΨm）驱动的Mito-proGreen在线粒体中的富集，以及捕获的探针与代谢整合至线粒体内膜的叠氮磷脂（azidophospholipids）之间的共价结合。当发生线粒体自噬时，ECAM会被转运至溶酶体，并被亮氨酰氨基肽酶（LNPEP/leucyl aminopeptidase）水解，产生可激活的绿色荧光信号；该信号不受溶酶体酸度变化的影响，且可在固定细胞中稳定留存。
借助ECAM探针，本研究证实佛波醇-12-肉豆蔻酸酯-13-乙酸酯（PMA）是一种强效线粒体自噬诱导剂。ECAM克服了pH探针信号易受酸度干扰、以及ΔΨm探针易从受胁迫线粒体中解离流失的缺陷，为线粒体自噬及线粒体自噬诱导型治疗药物的研究提供了极具应用价值的工具。